CN118634317B - Use of lactoferrin in combination with ergothioneine in the preparation of a medicament for the prevention and/or treatment of Alzheimer's disease - Google Patents

Abstract

The present invention provides the use of lactoferrin combined with ergothioneine in the preparation of a medicine for preventing and/or treating Alzheimer's disease, and belongs to the field of medicine. Compared with the use of lactoferrin or ergothioneine alone, the present invention combines lactoferrin and ergothioneine as a pharmacological substance in a specific ratio, and plays a synergistic effect, can reduce the cell damage caused by Aβ _25-35 , reduce p-Tau protein expression, reduce oxidative stress levels and regulate apoptosis, alleviate memory damage and cognitive dysfunction, can reduce Aβ deposition in mouse plasma, and provide a new option for clinical prevention or improvement of cognitive function.

Description

Use of lactoferrin combined with ergothioneine in the preparation of drugs for preventing and/or treating Alzheimer's disease

Technical Field

The invention belongs to the field of medicine, and particularly relates to use of lactoferrin combined with ergothioneine in preparing a medicine for preventing and/or treating Alzheimer's disease.

Background Art

Alzheimer’s disease (AD) is the most common neurodegenerative disease today and the main cause of dementia. Currently, there are more than 40 million AD patients worldwide, and this number is expected to exceed 130 million by 2050. In the later stages of AD development, patients will lose their cognitive and self-care abilities, become unable to live independently, and seriously damage the quality of life of patients and their families, placing a huge burden on families and society. The pathogenesis of AD is very complex and has not yet been fully clarified. The pathological changes involved are intricate, and pathological changes at different stages of development may affect each other, hindering the development of new drugs. So far, there is still no drug that can cure and prevent the progression of the disease, so research on the prevention and improvement of AD is imminent.

Lactoferrin (LF) is an iron-binding glycoprotein with a molecular weight of 80 kDa. It not only regulates the body's iron metabolism, but also plays an important role in the body's anti-inflammatory and anti-oxidation. Lactoferrin plays an important role in neurodevelopment, cognition and neuroprotection by increasing the expression of brain-derived neurotrophic factor in experimental animals and reducing neuronal loss and neuroinflammation. A randomized controlled trial showed that compared with standard treatment, lactoferrin capsules (250 mg/day for three months) improved cognitive symptoms, oxidative stress, inflammation, apoptosis, β-amyloid protein and tau protein pathology-related biomarkers in patients with mild to moderate AD.

Ergothioneine (EGT) is a sulfur-containing histidine derivative that is found in large quantities in mushrooms. It has significant antioxidant and neuroprotective effects and is widely used in many industries, including food, health products, cosmetics, and medicine. In the food industry, ergothioneine, as a natural antioxidant, is widely used in various functional foods; in the cosmetics industry, it is often added to anti-aging and whitening products to help improve skin conditions and delay aging. In the medical field, ergothioneine is used to intervene in the treatment of oxidative stress-related diseases due to its powerful antioxidant properties. It can also protect the brain from oxidative damage and neuroinflammation, target potential pathologies of neurodegeneration, such as mitochondrial dysfunction and toxic amyloid protein accumulation, and even promote neurogenesis, which suggests that it has potential application value in the prevention and treatment of neurodegenerative diseases.

The combination of lactoferrin and ergothioneine has not yet been used for the prevention and treatment of Alzheimer's disease.

Summary of the invention

The present invention provides use of lactoferrin combined with ergothioneine in preparing a medicine for preventing and/or treating Alzheimer's disease.

The present invention also provides the use of lactoferrin combined with ergothioneine in preparing food for assisting in improving memory function.

Furthermore, the mass ratio of lactoferrin to ergothioneine is 1:2-8.

Furthermore, the mass ratio of lactoferrin to ergothioneine is 1:2 or 1:8.

The present invention also provides a combined medicine for preventing and/or treating Alzheimer's disease, which contains lactoferrin and ergothioneine for simultaneous or separate administration.

Furthermore, the mass ratio of lactoferrin to ergothioneine is 1:2-8.

Furthermore, the mass ratio of lactoferrin to ergothioneine is 1:2 or 1:8.

The present invention also provides a composition for assisting in improving memory function, which is an oral preparation prepared by taking lactoferrin and ergothioneine as active ingredients and adding acceptable auxiliary materials; the oral preparation is a granule, powder, pill, capsule or solution.

Furthermore, the mass ratio of lactoferrin to ergothioneine is 1:2-8, preferably 1:2 or 1:8.

The present invention also provides a method for preparing the above composition, which comprises the following steps:

Weigh lactoferrin and ergothioneine according to the ratio, add acceptable excipients or auxiliary ingredients, mix well, and obtain the product.

The invention discloses a method for preparing a medicine for preventing and/or treating Alzheimer's disease in combination with ergothioneine, or a method for preparing a food for assisting in improving memory function. Experimental results show that compared with the use of lactoferrin or ergothioneine alone, the invention discloses a method for combining lactoferrin and ergothioneine in a specific ratio as a pharmacological substance, which can more significantly reduce cell damage caused by Aβ _25-35 , reduce p-Tau protein expression, reduce oxidative stress levels, regulate apoptosis, and reduce Aβ deposition in plasma, and has a synergistic effect in improving memory function and alleviating cognitive dysfunction, thereby providing a new option for clinical prevention and treatment of Alzheimer's disease.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Data graph showing the effects of different intervention methods on the survival rate of neurons induced by Aβ _25-35 .

Figure 2. Effects of different intervention methods on AD markers (A: protein bands of intracellular p-Tau and Tau proteins after treatment with different experimental groups; B: expression of intracellular p-Tau relative to Tau after treatment with different experimental groups).

Figure 3. Effects of different intervention methods on oxidative stress (ROS) (A: intracellular fluorescence expression after treatment with different experimental groups; B: relative expression of intracellular fluorescence after treatment with different experimental groups).

Figure 4. Effects of different intervention methods on changes in MDA activity in cells.

Figure 5. Effects of different intervention methods on changes in SOD activity in cells.

Figure 6. Effects of different interventions on cell apoptosis (A: protein bands of intracellular apoptosis-related proteins Cleaved-Caspase-3, Bax and Bcl-2 after different interventions; B: expression of Cleaved-Caspase-3 relative to β-actin after different interventions; C: expression of Bax relative to β-actin after different interventions; D: expression of Bcl-2 relative to β-actin after different interventions).

Figure 7. Effects of different intervention methods on mitochondrial membrane potential (A: schematic diagram of cell JC-1 staining fluorescence; B: results of mitochondrial membrane potential).

Figure 8. Effects of different intervention methods on the spatial short-term working memory ability of mice (A: total number of arm entries of mice; B: spontaneous alternation rate).

Figure 9. Effects of different intervention methods on the spatial memory ability of mice (A: schematic diagram of positioning and navigation in the water maze; B: experimental results of escape latency; C: the number of times the mouse crosses the platform in the target quadrant; D: the time the mouse stays in the target quadrant).

Figure 10. Effects of different intervention methods on the Aβ content in mouse plasma (A: Aβ _1-40 content in mouse plasma under different treatment methods; B: Aβ _1-42 content in mouse plasma under different treatment methods).

DETAILED DESCRIPTION

The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

Embodiment 1, composition of the present invention

Formula: Lactoferrin 50 mg, Ergothioneine 100 mg

Preparation method: Weigh lactoferrin and ergothioneine according to the ratio, and add excipients acceptable to medicines or foods to obtain the product.

Embodiment 2, composition of the present invention

Formula: Lactoferrin 50 mg, Ergothioneine 400 mg

Preparation method: Weigh lactoferrin and ergothioneine according to the ratio, and add excipients acceptable to medicines or foods to obtain the product.

The beneficial effects of the present invention are demonstrated by experimental examples below.

Experimental Example 1: Effect of lactoferrin combined with ergothioneine on the survival rate of neurons induced by Aβ _25-35

1. Experimental methods

To detect the effects of lactoferrin and ergothioneine and their combination on the survival rate of neural cells (N2a), cells in the exponential growth phase were selected, and after trypsinization, the cells were prepared into a single cell suspension with MEM complete medium, seeded into 96-well plates at a density of 1.0×10 ⁴ cells/well, and incubated overnight in a cell culture incubator (37°C, 5% CO ₂ ). After 24 hours of addition of 100 μL ergothioneine (160 μg/ml), 100 μL lactoferrin (80 μg/ml), and 100 μL of the combination of the two (2:1 group: ergothioneine 160 μg/ml, lactoferrin 80 μg/ml; 8:1 group: ergothioneine 640 μg/ml, lactoferrin 80 μg/ml) to each well of each group, the medium was discarded, and 100 μL 20 μmol/L Aβ _25-35 was added to stimulate the cells for 24 hours. Then discard the culture medium and add 1/10 volume of Cell Counting Kit-8 (CCK-8) directly to the cell culture medium, mix thoroughly, add 100 μL of mixed culture medium to each well, continue to culture in the cell culture incubator for 1-4 hours, read the 450nm light absorption value with a microplate reader, and calculate the cell activity.

2. Experimental results

The experimental results are shown in Figure 1. After 24 hours of Aβ _25-35 treatment of N2a cells, the cell survival rate was about 70%. Compared with the model group treated with Aβ _25-35 , both ergothioneine and lactoferrin treatments can increase cell survival rate ( P < 0.005), but the combined use of ergothioneine and lactoferrin significantly increased the survival rate of AD model cells compared with the single treatment group ( P < 0.05), and reduced cell death by about 20%, showing a good synergistic effect.

Experimental Example 2: Effect of lactoferrin combined with ergothioneine on AD markers

1. Experimental methods

Neurofibrillary tangles formed by abnormal hyperphosphorylation of intracellular Tau protein are one of the most important pathological features of AD. The effects of ergothioneine and lactoferrin intervention on the level of phosphorylated Tau (p-Tau) protein in model cells (i.e., N2a cells treated with Aβ _25-35 ) were detected by Western blot. N2a cells were inoculated and administered according to the operating procedures of Experimental Example 1. After drug intervention, the cell culture plate was placed on ice, IP lysis buffer containing PMSF and phosphatase inhibitors was added, and the cells were fully blown, and then the cells were collected into a centrifuge tube with a cell scraper and ice-bathed for 20 min to fully lyse the cells. The cells were centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was taken to obtain the total protein. The protein concentration was quantified using the BCA (bicinchoninic acid) method, and the expression of p-Tau and Tau proteins was detected by Western blot.

2. Experimental results

The experimental results are shown in Figure 2. Compared with the blank control group, the intracellular p-Tau protein level was significantly increased after 24 h of induction with 20 μmol/L Aβ _25-35 ( P <0.001), while compared with the model group, the p-Tau protein level was decreased after 24 h of pre-intervention with ergothioneine and lactoferrin ( P <0.01). Compared with the lactoferrin and ergothioneine treatment groups alone, the protein expression of the lactoferrin and ergothioneine combination was significantly reduced ( P <0.01), indicating that the combination can synergistically reduce the pathological markers of AD model cells.

Experimental Example 3: Effect of lactoferrin combined with ergothioneine on oxidative stress (ROS)

1. Experimental methods

N2a cells were inoculated and dosed according to the operating procedures of Experimental Example 1. 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol/L. The cell culture medium was removed and an appropriate volume of the diluted DCFH-DA fluorescent probe was added so that the added volume could fully cover the cells. Incubate in a 37ºC cell culture incubator for 20 minutes and wash the cells three times with PBS buffer to fully remove the DCFH-DA fluorescent probe that did not enter the cells. Finally, a fluorescence microscope was used for observation and photography.

2. Experimental results

The experimental results are shown in Figure 3. ROS is an important sign of cellular oxidative damage. After Aβ _25-35 treatment, the intracellular ROS level was significantly higher than that of the blank control group ( P <0.001), indicating that excessive ROS has been produced in the cells, the body's antioxidant defense system cannot effectively remove ROS, and the oxidation and antioxidant effects are unbalanced, which in turn causes cellular oxidative damage. Both ergothioneine and lactoferrin treatment significantly reduced the level of ROS in cells ( P <0.01), and the combined use of ergothioneine and lactoferrin reduced ROS in cells by more than 40% compared with the Aβ model group, and was significantly lower than the single treatment group ( P <0.01), indicating that the combined use of lactoferrin and ergothioneine can synergistically improve cellular oxidative damage.

Experimental Example 4: Effect of lactoferrin combined with ergothioneine on oxidative stress products (MDA and SOD)

1. Experimental methods

The content of malondialdehyde (MDA) is an important parameter reflecting cell damage and the body's antioxidant potential. Superoxide dismutase (SOD) is a key enzyme for preventing cell oxidative stress damage. By measuring the effects of ergothioneine and lactoferrin on the activity of cell MDA and SOD, its intracellular antioxidant capacity can be determined. According to the instructions of the MDA and SOD kits, the optical density (OD) value of the sample was detected using a microplate reader, where the sample was prepared by the inoculation and administration method according to the operating procedures of Experimental Example 1. The content of intracellular MDA and SOD was calculated according to the instructions.

2. Experimental results

The results (Figure 4) showed that compared with the blank control group, Aβ _25-35 treatment significantly increased the intracellular MDA content ( P <0.001), the MDA content in the ergothioneine and lactoferrin groups decreased by 0.36 and 0.4 nmol/mg, respectively ( P <0.01), and the MDA content in the combination of ergothioneine and lactoferrin decreased by more than 0.6 nmol/mg, which was significantly lower than that in the single treatment group ( P <0.05).

The changes in SOD activity in cells are shown in Figure 5. Under the induction of Aβ _25-35 , the activity of SOD was significantly lower than that of the control group ( P < 0.001). Compared with the Aβ treatment group, the SOD activity of the ergothioneine and lactoferrin groups increased by more than 0.2 mU/mg, and the difference was statistically significant ( P < 0.05), while the SOD activity of ergothioneine combined with lactoferrin increased by more than 0.3 mU/mg, which was significantly higher than that of the single treatment group ( P < 0.05), indicating that the combination of ergothioneine and lactoferrin can synergistically improve the antioxidant capacity of cells.

Experimental Example 5: Effect of lactoferrin combined with ergothioneine on cell apoptosis

1. Experimental methods

Aβ can promote cell apoptosis by regulating the expression of apoptosis-related proteins. Western blot was used to detect the effects of different intervention methods on the expression of apoptosis-related proteins Cleaved-Caspase-3, Bax and Bcl-2 in cells. The specific samples were prepared according to the inoculation and administration methods of the operating procedures of Experimental Example 1.

2. Experimental results

The results (Figure 6) showed that compared with the blank control group, after 24 h of induction with 20 μmol/L Aβ _25-35 , the expression of pro-apoptotic proteins Cleaved-Caspase-3 ( P <0.005) and Bax ( P <0.001) in the cells increased, while the expression of anti-apoptotic protein Bcl-2 decreased ( P <0.001). Compared with the model group, after 24 h of intervention with ergothioneine and lactoferrin and their combination, the expression of Cleaved-Caspase-3 ( P <0.01) and Bax ( P <0.05) proteins in the cells decreased, and the expression of Bcl-2 increased significantly ( P <0.05). Among them, the expression of pro-apoptotic proteins in ergothioneine and lactoferrin was reduced compared with the single treatment group, while the expression of anti-apoptotic proteins was increased ( P <0.05), indicating that lactoferrin and ergothioneine can synergistically inhibit cell apoptosis.

Experimental Example 6: Effect of lactoferrin combined with ergothioneine on mitochondrial membrane potential

1. Experimental methods

In the early stage of apoptosis, the mitochondrial transmembrane potential decreases, which leads to mitochondrial dysfunction, ATP synthesis disorder, ROS accumulation, and oxidative stress. Therefore, detecting the changes in mitochondrial membrane potential is one of the effective means to determine whether cells have undergone early apoptosis. Take an appropriate amount of mitochondrial membrane potential detection reagent (JC-1, 200X) and dilute JC-1 at a ratio of 8ml ultrapure water per 50µl JC-1 (200X). Vortex vigorously to fully dissolve and mix JC-1. Then add 2ml JC-1 staining buffer (5X), and mix to obtain JC-1 staining working solution. Add 0.5ml cell culture medium to the 12-well plate, then add 0.5ml JC-1 staining working solution, and mix thoroughly. Incubate at 37ºC in a cell culture incubator for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) at a ratio of 4ml distilled water per 1ml JC-1 staining buffer (5X), and place it in an ice bath. After incubation at 37°C, remove the supernatant, wash twice with JC-1 staining buffer (1X), and add 1 ml of cell culture medium. Observe and photograph under a fluorescence microscope.

2. Experimental results

The staining results (Figure 7) showed that compared with the blank control group, after 24 h of induction with 20 μmol/L Aβ _25-35 , a large amount of JC-1 in the matrix existed in monomeric form, producing a large amount of green fluorescence ( P <0.001). Compared with the model group, after 24 h of pre-intervention with ergothioneine and lactoferrin, the green fluorescence intensity of the cells decreased and the red fluorescence intensity increased ( P <0.05), indicating that ergothioneine and lactoferrin can effectively alleviate the decrease in cell mitochondrial membrane potential induced by Aβ _25-35 . After the combined use of the two, the membrane potential was further reduced ( P <0.05), indicating that the combination of ergothioneine and lactoferrin can further alleviate early cell apoptosis.

Experimental Example 7: Effects of lactoferrin combined with ergothioneine on spatial short-term working memory ability of APP/PS1 mice

1. Experimental methods

(1) Animal grouping and treatment methods: 40 5-month-old APP/PS1 mice and 8 wild-type mice of their littermates were purchased from Beijing Zhishan Health Medical Research Institute Co., Ltd. After all mice were adaptively fed for 1 week, the APP/PS1 mice were randomly divided into 5 groups, namely the model group (Saline), EGT group, LF group, EGT+LF (2:1) group, and EGT+LF (8:1) group, with 8 mice in each group. Wild-type mice served as the blank control group (Ctrl). The LF group was given 50 mg/kg/d of exogenous lactoferrin, and the EGT group was given 100 mg/kg/d. The EGT+LF (2:1) group was given 50 mg/kg/d of exogenous lactoferrin and 100 mg/kg/d of ergothioneine. The EGT+LF (8:1) group was given 50 mg/kg/d of exogenous lactoferrin and 400 mg/kg/d of ergothioneine. The model group and the blank control group were given the same dose of normal saline. Each group was treated by gavage for 90 days.

(2) Detection method: The Y-maze is used to detect the spatial short-term working memory ability of animals. The box of the Y-maze experiment consists of three arms and a connecting area. The angle between the three arms is 120°, and each arm is 30 cm long. After placing the tested mouse at the end of an arm, let the mouse freely explore the three arms of the Y-maze for 5 minutes. The number of times and total distance of the mouse in each arm are detected. When the center point of the mouse is located in the arm, it is considered that the mouse has entered the arm. After each test, the instrument is cleaned with 75% ethanol and dried thoroughly. Indicator: Spontaneous alternation percentage (%) = [number of alternations/(total number of entries into each arm-2)] × 100%.

2. Experimental results

The results are shown in Table 1 and Figure 8. As shown in Figure 8, in the Y-maze spontaneous alternation test, there was no significant difference in the total number of arm entries among the mice in each group (P>0.05) (Figure 8A), indicating that there was no difference in the motor ability of the mice in each group. However, compared with wild-type mice (see Table 1), the accuracy of spontaneous alternation in the APP/PS1 + Saline group was reduced by 46% ( P <0.001), and the accuracy of spontaneous alternation in the model mice increased by more than 15% after intervention with lactoferrin or ergothioneine ( P <0.05). The combined use of lactoferrin and ergothioneine increased the accuracy of spontaneous alternation by at least 21% compared with the single treatment group, and the difference was statistically significant ( P <0.05) (Figure 8B), suggesting that the combined use of lactoferrin and ergothioneine can better alleviate the impairment of working memory in AD model mice.

Table 1. Spontaneous alternation rate of different experimental groups after treatment

Further comparison of the percentage of alternation rate increase after lactoferrin, ergothioneine, and the combined use of lactoferrin and ergothioneine relative to the Saline group in Table 1 showed that the alternation rate improvement effect after the combined use of lactoferrin and ergothioneine (2:1 group: 41%, 8:1 group: 39%) was higher than the sum of the alternation rate improvement effects after lactoferrin alone and ergothioneine alone (33%), indicating that the combined use of lactoferrin and ergothioneine played a synergistic effect in improving the spontaneous alternation rate.

Experimental Example 8: Effects of lactoferrin combined with ergothioneine on spatial memory ability of APP/PS1 mice

1. Experimental methods

The Morris water maze test is used to evaluate the spatial learning and reference memory abilities of animals. The water maze body is a round barrel structure with a diameter of 120 cm and a height of 50 cm. Before the experiment, an appropriate amount of water was injected into the body, and an appropriate amount of titanium dioxide was added to the water to mix well, making the water milky white, so that the camera and recording system can record and analyze the movement trajectory of the mice. The water temperature was kept at 22-26°C throughout the experiment.

The inside of the uterus was divided into four quadrants by computer software, and the escape platform was hidden about 1 cm below the water surface in the target quadrant. Four black marks of different shapes were made on the inner wall of the uterus in the four quadrants above the water surface to facilitate the mice to find the platform. The experimental contents mainly included positioning navigation experiment and space exploration experiment, which were used to test the spatial learning ability and spatial reference memory ability of mice respectively.

In the navigation experiment, mice were randomly placed in the water from one of the quadrants facing the pool wall. When the mice found the platform, they were allowed to stay on the platform for 10 seconds. If they did not find the platform within the prescribed 60 seconds, they were guided to the platform and stayed for 10 seconds. The training was conducted 4 times a day, once in each quadrant, with an interval of less than 20 minutes each time. The experiment lasted for 4 days, during which the escape latency of the mice to find the underwater platform was mainly observed.

On the 5th day, a spatial exploration experiment was conducted, during which the platform was removed and a quadrant other than the target quadrant was selected. The mice were placed in a pool and allowed to swim freely for 1 min. The percentage of the swimming time in the target quadrant to the total swimming time and the number of times the mice crossed the platform were recorded to evaluate the memory ability of the mice.

The specific experimental animal grouping and treatment methods were the same as those in Experimental Example 7.

2. Experimental results

The results of the navigation experiment in the water maze (Figure 9) showed that the escape latency of the APP/PS1 + Saline group mice on the 4th day was prolonged by 26.6s (P<0.001) compared with the wild-type control group mice, indicating that the APP/PS1 mice of this age had already shown spatial learning impairment; after intervention with lactoferrin and ergothioneine, the escape latency of the model mice was shortened by more than 9.8s on the 4th day (P<0.001). Compared with the single treatment group, the lactoferrin and ergothioneine combination group can significantly shorten the latency time (P<0.05) (Figures 9A and 9B), indicating that the lactoferrin and ergothioneine combination can synergistically alleviate the spatial learning impairment of APP/PS1 mice. In the spatial exploration experiment, the APP/PS1+Saline group, i.e., the model group, had significantly less time spent in the target quadrant and more times of crossing the platform than the wild-type control group (P<0.001). However, after the combined use of lactoferrin and ergothioneine, the mice had significantly more time spent in the target quadrant and more times of crossing the platform than the model group (P<0.001), and were stronger than the group using lactoferrin and ergothioneine alone (P<0.05) (Figures 9C and 9D). This result indicates that the combination of lactoferrin and ergothioneine can improve the spatial reference memory ability of APP/PS1 mice and alleviate cognitive dysfunction.

Experimental Example 9: Effect of lactoferrin combined with ergothioneine on plasma Aβ content in APP/PS1 mice

1. Experimental methods

The blood of the mice in each group after treatment in Experimental Example 7 was collected and centrifuged at 1000×g for 15 minutes at 4° C. The supernatant was taken and the levels of Aβ _1-40 and Aβ _1-42 in the plasma were detected using an ELISA kit.

2. Experimental results

The results (Figure 10) showed that compared with the wild-type control group mice, the levels of Aβ _1-40 and Aβ 1-42 in the plasma of the APP/PS1 + Saline group mice increased by 313 pg/mL and 188 pg/mL, respectively ( P <0.001), and the difference was statistically significant; the levels of Aβ _1-40 and Aβ _1-42 in the APP/PS1 mice fed with lactoferrin and _{ergothioneine} were reduced by 169 pg/mL and 191 pg/mL, respectively, compared with the Saline group ( P <0.05). The Aβ _1-40 content of the lactoferrin combined with ergothioneine group was significantly lower than that of the single-use group ( P <0.05), and the Aβ _1-42 content of the combined use group with a ratio of 8:1 was slightly reduced, but the difference was not statistically significant ( P ＞ 0.05). This indicates that lactoferrin combined with ergothioneine significantly reduces the plasma Aβ content of AD model mice.

From the above experimental results, it can be seen that the combined use of lactoferrin and ergothioneine can reduce the cell damage caused by Aβ _25-35 , reduce the expression of p-Tau protein, reduce the level of oxidative stress, regulate apoptosis, alleviate memory impairment and cognitive dysfunction, and reduce Aβ deposition in mouse plasma.

In summary, the present invention provides the use of lactoferrin combined with ergothioneine in the preparation of a medicament for preventing and/or treating Alzheimer's disease. Experimental results show that, compared with the use of lactoferrin or ergothioneine alone, the present invention combines lactoferrin and ergothioneine in a specific ratio as a pharmacological substance, which has a synergistic effect in improving memory function and alleviating cognitive dysfunction, and provides a new option for clinical prevention or treatment of Alzheimer's disease.

Claims (8)

1. Use of lactoferrin in combination with ergothioneine in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the mass ratio of the lactoferrin to the ergothioneine is 1:2 to 8.

2. Use of lactoferrin in combination with ergothioneine for the preparation of a food product for aiding in improving memory function, characterized in that: the mass ratio of the lactoferrin to the ergothioneine is 1:2 to 8.

3. Use according to claim 1 or 2, characterized in that: the mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:8.

4. A combination medicament for treating alzheimer's disease, characterized in that: it contains lactoferrin and ergothioneine for simultaneous or separate administration; the mass ratio of the lactoferrin to the ergothioneine is 1:2 to 8..

5. The combination according to claim 4, wherein: the mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:8.

6. A composition for aiding in improving memory, characterized in that: the preparation is prepared by adding acceptable auxiliary materials into lactoferrin and ergothioneine serving as active ingredients; the mass ratio of the lactoferrin to the ergothioneine is 1:2 to 8..

7. The composition of claim 6, wherein: the mass ratio of the lactoferrin to the ergothioneine is 1:2 or 1:8, 8;

and/or, the formulation is an oral formulation; the oral preparation is granule, powder, pill, capsule or solution.

8. A process for the preparation of a composition as claimed in claim 6 or 7, characterized in that: it comprises the following steps:

weighing lactoferrin and ergothioneine according to the proportion, adding acceptable auxiliary materials or auxiliary components, and uniformly mixing.